Project Summary The purpose of each ENCODE Functional Characterization Center is ?to develop and apply generalizable approaches to characterize the role of candidate functional elements identified the ENCODE project in specific biological contexts?. Our proposed Center will focus on characterization of candidate enhancer elements. We will develop, refine and apply experimental methods for functional assays of enhancers. We will use two very different biological models chosen for their high potential to act as generalizable exemplars for the study of enhancers in the context of (i) inherited risk factors for disease, and (ii) somatic mutations involved in cancers. We will also develop and refine our experimental methods in ENCODE cell lines, and we will reserve 25% of our efforts for testing candidate genomic elements that will be studied in common across all of the ENCODE Functional Characterization Centers. Using STARR-seq, and variations thereof, we will test for sufficiency of candidate enhancer elements to modulate gene expression. Using CRISPR-Cas9 methods we will edit the human genome, testing for necessity of candidate enhancer elements in their endogenous context. We will utilize these methods to examine the effects of inherited DNA variation on enhancer function in models of coronary artery disease (CAD), and to examine the effects of acquired somatic DNA mutations on enhancer function in models of pancreatic cancer (Pancreatic Ductal Adenocarcinoma ? PDAC). While our approach necessarily requires a bioinformatics component to utilize ENCODE and other existing data sets in order to define the best candidate enhancer elements for testing in the specific biological models we will assay, our Center will be focused on experimental characterization of enhancers, testing different combinations of approaches in order to create extensible and generalizable protocols for systematic and accurate characterization of enhancer function.